cGMP-dependent protein kinase (PKG) is the central enzyme of NO-cGMP signaling pathway that regulates platelet aggregation, smooth muscle tone, phototransduction, and leukocyte migration [1,2]. Although PKG has been heavily targeted for treating diseases such as, erectile dysfunction and cardiovascular and pulmonary diseases, developing specific activators and inhibitors has been difficult because there is no structural information available[1,2]. For the successful NO-cGMP mediated signaling responses to occur, PKG has to be localized to the specific site in the cell. Localization of PKG is an essential feature of the NO-cGMP signaling and mediated by G Kinase Anchoring Proteins (GKAPs)[3,4,5,6,7,8]. In particular, the variable leucine zipper domain at the extreme N-terminus targets PKG to specific subcellular sites via its interaction with G-kinase anchoring proteins in an isotype specific manner [7,9]. Despite mounting evidence that GKAP target PKGs via its association with the zipper domain, the molecular details of this important protein- protein interaction remain unknown. The specific aims of this proposal are to understand the isotype specific targeting mechanism of PKGs using peptide arrays in combination with X-ray crystallography. Adding another layer of complexity, there are three types of PKG (I1, I2, and II) that each have different cGMP dependence in activation, unique sets of substrates, and tissue specific expression [2,10,11]. My primary focus is the type I isozymes (1 and 2) that have been implicated in erectile dysfunction and many cardiovascular diseases. Although they represent functionally non- redundant proteins with unique physiological roles, their functional roles and specific subcellular localization are poorly understood. In order to elucidate isozyme specific functions and localization, I plan to solve crystal structures of the PKG I1 and 2 zipper domains and compare molecular features of GKAP docking surfaces that are unique to each isoform. In parallel, I plan to engineer peptides that can selectively disrupt interactions between both isozymes of PKG I and their individual binding partners. Lastly, I will incorporate high affinity peptides into the co-crystallization trials in order to understand molecular details of the PKG/GKAP interaction. My plan is to form stable protein/peptide complexes using isozyme specific peptides and pursue high-resolution crystal structures of PKG/GAKP complexes. Solution of the zipper domain structures, and development of small peptides that specifically disrupt isozyme specific targeting, will pave the way to elucidation of the specific functions of PKGs and eventually lead to the development of therapeutic agents. PUBLIC HEALTH RELEVANCE: cGMP-dependent protein kinase (PKG) is the central enzyme of NO-cGMP signaling pathway that regulates platelet aggregation, smooth muscle tone, phototransduction, and leukocyte migration. Although PKG has been heavily targeted for treating diseases such as, erectile dysfunction and cardiovascular and pulmonary diseases, developing specific activators and inhibitors has been difficult because there is no structural information available. My ultimate goal is to rationally target the kinase by obtaining high-resolution crystal structures of PKG and its isozymes and develop pharmacological agents that can modulate the activity of the kinase to treat diseases related to NO-cGMP signaling dysfunction.